Car t-cells for the treatment of cd1a-positive cancer

ABSTRACT

Relapsed/refractory T-cell acute lymphoblastic leukemia (T-ALL) has a dismal outcome, and no effective targeted immunotherapies for T-ALL exist. The extension of chimeric antigen receptor T-cells (CARTs) to T-ALL remains challenging because the shared expression of target antigens between CARTs and T-ALL blasts leads to CARTs fratricide. CD1a is exclusively expressed in cortical T-ALLs, a major subset of T-ALL. The expression of CD1a is restricted to cortical thymocytes and neither CD34+ progenitors nor T-cells express CD1a during ontogeny, confining the risk of on-target/off-tumor toxicity. The present invention provides CARs comprising a CD 1a-targeting moiety which may be transduced or transformed into T cells. The resultant CARTs are suitable for the treatment of cortical T-ALLs.

TECHNICAL FIELD

The present invention provides therapeutics for the treatment ofCD1a-positive cancers such as T-cell acute lymphoblastic leukemia andT-cell lymphoblastic lymphoma. In particular, the present inventionprovides chimeric antigen receptor (CAR) T-cells that can target CD1a.

BACKGROUND ART

T-cell lineage acute lymphoblastic leukemia (T-ALL) is a malignantdisorder resulting from leukemic transformation of thymic T-cellprecursors¹. T-ALL is phenotypically and genetically heterogeneous, andis commonly associated with genetic alterations/mutations intranscription factors involved in hematopoietic stem/progenitor cell(HSPC) homeostasis and in master regulators of T-cell development².T-ALL comprises 10-15% and 20-25% of all acute leukemias diagnosed inchildren and adults, respectively^(3,4) with a median diagnostic age of9 years⁵⁻⁷. Intensive chemotherapy regimens have led to the improvedsurvival of patients with T-ALL. However, the event-free (EFS) andoverall (OS) survival remains <70%, and relapsed/refractory (R/R) T-ALLhas a particularly poor outcome. There are currently no potentialcurative options beyond hematopoietic cell transplantation andconventional chemotherapy, which is linked to large trade-offs intoxicities^(4,8), reinforcing the need for novel targeted therapies.T-cell lymphoblastic lymphomas (TCL) are etiologically andpathogenically different from T-ALL but phenotypically very similar. Themain difference is that TCLs are found extramedullary while T-ALL is abone marrow disease.

Immunotherapy has generated unprecedented expectations in cancertreatment and relies on the immune system as a powerful weapon againstcancer. In recent years, adoptive cellular immunotherapy based onchimeric antigen receptors (CARs) has shown great potential. CAR therapyredirects genetically modified T-cells to specifically recognize andeliminate specific antigen-expressing tumor cells in a majorhistocompatibility complex-independent fashion^(9,10). The success ofCAR T-cells (CARTs) redirected against CD19 or CD22 is now indisputablefor B-cell malignancies (mainly B-ALL)^(11-14.) But, strategiestargeting T-cell malignancies using CARTs remain challenging because ofthe shared expression of target antigens between CARTs and T-lineagetumoral cells. In this regard, CARTs against pan T-cell antigens havetwo major drawbacks: i) CARTs self-targeting/fratricide and, ii) T-cellaplasia, leading to life-threating immunodeficiency¹⁵⁻¹⁷.

Recent elegant studies demonstrated that T-cells transduced with eitherCD7, CD3, CD5 or TCR CARS, the most expressed pan-T-cell antigens,efficiently eliminate T-ALL blasts in vitro and are able to editing orprotein expression blockers, were required for disruption of the targetantigen in T-cells prior to CAR transduction, to avoid extensiveself-antigen driven fratricide^(15-17,19).

Thus, there remains a need for a therapy that can successfully treatT-ALL. The present invention aims to provide a therapy for treatingCD1a-positive T-ALL.

FIGURES

FIG. 1. CD1a expression in T-ALL and normal hematopoiesis andthymopoiesis. (A) Immunophenotype of de novo T-ALL samples (n=38) forthe indicated markers. Upper and intermediate curly brackets identifyCD1a^(+/++) and CD1a^(low/+) coT-ALL patients, respectively. Blackcircles at bottom depict non-coTALL patients. (B) Representative FACSdot plot of a coT-ALL patient. CD7+CD1a+ cells are coT-ALL blasts andCD3+CD7+CD1a− (either CD4+ or CD8+) are normal mature T-cell present inthe diagnostic sample. (C) CD1a is retained at relapse (n=5diagnostic-relapse coT-ALL pairs). Data shown as CD1a expression inrelapse samples relative to the diagnostic-matched samples (diagnosticshown as 100% expression). (D) T-cells and CD34+ HSPCs do not expressCD1a across ontogeny. (E) Scheme depicting the phenotype of developingthymic T-cell populations. (F) Representative FACS for pre-cortical(CD34^(high)CD7++CD1a−) and cortical (CD34+CD7++CD1a+) thymocytes. DX:diagnostic. RX: relapse.

FIG. 2. CD1a CARTs specifically target and eliminate CD1a+ T-ALL celllines in vitro. (A) Scheme of the CD1aCAR construct used. (B) CARdetection in 293T cells using an anti-scFv MoAb and GFP. (C)Representative CAR transduction and detection in CD4+ and CD8+ T-cells(n=6). (D) Proper T-cell activation (n=3). (E) Robust expansion ofactivated T-cells transduced with either mock or CD1a CAR reveals nosigns of fratricide (n=4). (F) Surface expression of CD1a (black line)in Jurkat, MOLT4 and NALM6 cell lines. (G) CD1a antigen density in celllines, primary coT-ALL samples and primografts. (H) Cytotoxicity of CD1aCARTs and MOCK T-cells against coT-ALL and B-ALL cell lines at theindicated E:T ratios in 16 h assays (n=4). (I) Absolute counts of aliveeFluor+ target cells measured by FACS in 72 h cytotoxicity assays at 1:1E:T ratio. (J) Representative FACS analysis of cytotoxicity with targetcells labeled with eFluor670. (K) ELISA showing high-level production ofthe inflammatory cytokines IL-2, TNFα and IFNγ by CD1a CARTs exposed toJurkat and NALM6 (negative control) cells in 16 h assays at 1:1 E:Tratio (n=4). *p<0.05, **p<0.01, ***p<0.001.

FIG. 3. CD1a CARTs specifically target and eliminate in vitro CD1a+T-ALL blasts from primary samples or PDX models. (A) Expression of CD1avs CD7 in coT-ALL blasts from primary patients/primografts. The % ofCD1a+ blasts is indicated. (B) Cytotoxicity (in absolute counts ofeFluor+ cells) measured by FACS in 48 h cytotoxicity assays at 4:1 E:Tratio (n=3). (C) Representative FACS analysis of CD1a within theeFluor-labeled target cells at the end of the cytotoxicity assay,revealing specificity of CD1a CARTs (n=3). (D) High-level production ofpro-inflammatory cytokines by CD1a CARTs analyzed by ELISA (n=3independent supernatants) in 16 h assays at 4:1 E:T ratio. *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001.

FIG. 4. CD1a CARTs fully control the progression of coT-ALL cells in amouse xenograft setting. (A) Scheme of the xenograft model. NSG mice(n=6/group) were i.v. injected with 3×10⁶ Luc-GFP-expressing Jurkatcells followed 3 days after by a single i.v. injection of 5×10⁶ mock orCD1a CARTs. Tumor burden was monitored every 4-6 days by bioluminescence(BLI) using IVIS imaging. When MOCK-treated animals were fully leukemic,one-half of the CD1a CARTs-treated animals were sacrificed and analyzedby FACS (BM, PB and spleen) for leukemic burden and CARTs persistence.The remaining animals were re-challenged 6 weeks after with 1.5×10⁶Luc-Jurkat and were followed-up as before. (B) IVIS imaging of tumorburden monitored by BLI at the indicated timepoints. (C) Total radiancequantification (p/sec/cm²/sr) at the indicated timepoints. †: sacrifice.(D) Circulating Jurkat cells in PB 17 days after CARTs infusion. (E)T-cell persistence in PB at day 17, and spleen and BM at sacrifice. Datais shown as mean±SD (n=6 mice/group). *p<0.05, **p<0.01, ***p<0.001.

FIG. 5. CD1a CARTs fully abolish the progression of primary CD1a+coT-ALL blasts in a PDX setting. (A) Scheme of the PDX model. NSG mice(n=5-6/group) were i.v. injected with 1×10⁶ primary coT-ALL cellsfollowed three days after by a single i.v. injection of 1×10⁶ mock orCD1a CARTs. Tumor burden was monitored by FACS every other week bybleeding and BM aspirate after 6 and 9 weeks. (B,C) Frequency ofleukemic mice and levels of leukemia in BM (B) and PB (C) 6 and 9 weeksafter infusion of CARTs. The left panels show representative FACS plots.Primary CD1a+ T-ALL blasts are shown inside the box (grey). EffectorT-cells are shown outside the box in grey. Mouse cells are shown inblack. (D) 9-week OS of coT-ALL primografts receiving either CD1a CARTsor MOCK T-cells. (E) Effector T-cell persistence overtime in PB (week 2towards week 9) and BM (week 6 and 9). Each dot represents anindependent mouse. **p<0.01, Malcolm-Cox test.

FIG. 6. CD1a CARTs retain the ability to control progression of CD1a+cell lines and coT-ALL primary samples in a re-challenge PDX setting.(A) IVIS imaging of Jurkat cells burden in the re-challenged mice. (B)Total radiance quantification (p/sec/cm²/sr) overtime in the micere-challenged with Jurkat cells. (C) Circulating Jurkat cells in PB 16days after re-challenge. (D) Robust effector T-cell persistence in PB,BM and spleen at sacrifice of the re-challenged animals. (E) Scheme ofthe re-challenge PDX experiments using coT-ALL primary samples.CARTs-bearing PDX mice were re-challenged with 1×10⁶ primary CD1a+ T-ALLseven weeks after initial CARTs infusion. (F) Secondary coT-ALL burdenin engrafted PB (left panel) and BM (right panel) 6 weeks after leukemiare-challenge. (G) Effector T-cell persistence overtime in PB (week 2, 4and 6) from PDXs re-challenged with coT-ALL primary samples. Each dotrepresents an independent mouse. *p<0.05, **p<0.01, ***p<0.001,****<0.0001.

FIG. 7. CD1a CARTs derived from coT-ALL patients at presentationspecifically lyse autologous CD1a+ T-ALL blasts. (A) Scheme depictingthe experimental design for the autologous cytotoxic assay. Mature(normal) CD3+CD1a⁻ T-cells were FACS-purified from the PB of a coT-ALLpatient, infected with CD1a CAR, expanded, and exposed to autologoustotal PBMCs. (B) FACS analysis of autologous cytotoxic 48 h-assay at 1:1and 4:1 E:T. eFluor670-labeled total PBMC target population containsCD1a+ T-ALL blasts (upper box) and mature CD3+CD1a− T-cells (bottombox). (C) Quantification of CD1a CARTs-mediated specific lysis forcoT-ALL blasts (upper panel) and CD3+CD1a− mature T-cells (bottompanel). (D) ELISpot showing the number of IFNγ SFC from mock versus CD1aCARTs stimulated with a pool of peptides from CMV, EBV and Flu (CEF).Staphylococcal enterotoxin B (SEB) was used as positive control.

FIG. 8. Immunophenotype for each individual CD1a++ coT-ALL patientpresented in this study. (A) Gating strategy distinguishing maturenormal T-cells (CD3++CD1a− either CD4+ or CD8+) and coT-ALLs blasts(CD7+CD1a+). Note that coT-ALL blasts commonly have aberrant expressionfor CD3 and/or CD4/CD8). (B) CD7/CD3 vs CD1a FACS dot plots for n=16available CD1a++ coT-ALL patients showing the percentage of maturenormal T-cells (left quadrant) and coT-ALLs blasts (right quadrant).

FIG. 9. In vitro specificity of CD1a CARTs. (A) Scheme of the CD1aCAR,CD22CAR and MOCK constructs used in the present study. (B) CD1a CARTsbut not CD22 CARTs lyse the T-ALL cell line Jurkat. CD22 CARTs but notCD1a CARTs lyse the B-ALL line NALM6. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.

FIG. 10. In vivo cytotoxicity of CD1a CARTs is dose-dependent. (A) Tumorburden monitored by BLI at the beginning of the experiment (scale: 3×10⁴to 1×10⁵ p/sec/cm²/sr) confirming early and efficient T-ALL engraftment.(B) IVIS imaging of tumor burden monitored by BLI at the indicated timepoints for CARTs doses of 2×10⁶ and 5×10⁶ p/sec/cm²/sr. (C) Totalradiance quantification (p/sec/cm²/sr) at the indicated time points forCARTs doses of 2×10⁶ and 5×10⁶. N=3-4 mice/group. †: sacrifice. *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001.

FIG. 11. CD1a CARTs do not target CD7+CD1a− thymocytes. Cytotoxicityassays against fetal thymic cells were performed at 16 h and 72 h at 4:1E:T for CD1a CARTs and MOCK T-cells (n=2).

FIG. 12. The absolute number of CD1a− primary coT-ALL cells remainsidentical after either MOCK or CD1a CART exposure. This confirms thatCD1a expression was not lost/downregulated by immune pressure.

SUMMARY OF THE INVENTION

The choice of the antigen against which we wish to re-direct T-cellsrepresents a major advance to solve the problems associated with theshared expression of T-cell markers between normal and malignantT-cells. We identified that CD1a, a lipid-presenting molecule, is asuitable target for treating a large subset of T-ALL, i.e. corticalT-ALL.

We developed and functionally characterized CD1a-specific CARTs, whichdisplayed robust cytotoxicity against T-ALL cell lines and primarycortical CD1a+ T-ALL cells both in vitro and in vivo in xenograftmodels. The CD1a CARTs continuously expanded 200-fold, similar to MOCKT-cells, demonstrating that redirecting CARTs against CD1a antigen doesnot induce T-cell fratricide. Also, the use of CD1a CARTs for corticalT-ALL bypasses the need for sophisticated genome editing-baseddisruption of target antigens in T-cells prior to CAR transduction as astrategy to avoid self-antigen-driven fratricide^(15-17,19). We furtherdemonstrated that in steady-state hematopoiesis, CD1a is exclusivelyexpressed in a subset of cortical CD34+CD7+ thymic T progenitors,whereas earlier CD34^(high)CD7^(high) T-progenitors lack CD1a. Inaddition, neither normal CD34+ HSPCs nor mature T-cells from multipletissues express CD1a during ontogeny, thereby minimizing the risk ofon-target/off-tumor toxicity. Indeed, when human fetal thymus-derivedCD7+ thymocytes were exposed to CD1a CARTs, only the CD1a+ corticalthymocytes were eliminated by the CD1a CARTs, while developmentallyearlier and later thymic T-lineage populations (CD34+ and CD34−) werenot targeted, limiting the on-target/off-tumor effects to adevelopmentally transient thymic population of cortical thymocytes andfurther confirming the fratricide resistant nature of CD1a CARTs.

The exclusive thymic localization of cortical thymocytes, and the factthat thymic subpopulations of CD34+CD7+CD1a− T-cell progenitorsphysiologically/constantly maturing into functional T cells resideupstream of CD1a+ cortical thymocytes, provides an additional level ofsafety for the use of CD CARTs in patients with R/R T-ALL. We do notexpect irreversible toxicities or severe T-cell aplasia attributed toCD1a CARTs for the following reasons: i) the CD1a+ thymocyte populationis a transient thymic T-cell fraction, eventually regenerated byupstream CD1a− T-cell progenitors; ii) CD1a CARTs themselves respondnormally to viral antigens and therefore are likely to be protectiveagainst pathogens; iii) the clinical use of specific antibodies againstCD5 or CD7⁴² did not reveal severe or irreversible toxicities; iv) thereare multiple studies that demonstrate extrathymic maturation of T-cellsand a balance between the innate and adaptive immune system that may, atleast in part, guarantee immunological protection in patients who haveundergone partial or total thymectomy⁴⁵⁻⁴⁷.

Thus, in one aspect, the present invention provides a chimeric antigenreceptor (CAR) comprising an extracellular domain comprising a CD1atargeting-moiety, a transmembrane domain, and an intracellular signalingdomain.

The present invention also provides a nucleic acid encoding the CAR ofthe present invention. Further, the present invention provides a cellcomprising the nucleic acid and/or CAR of the present invention. And,the present invention provides a pharmaceutical composition comprising aplurality of cells in accordance with the present invention and apharmaceutically acceptable carrier or diluent.

The cell of the present invention or pharmaceutical composition of thepresent invention is provided for use as a medicament. In particular,the present invention provides a method of treating a CD1a-positivecancer comprising administering the cell of the present invention or thepharmaceutical composition of the present invention to a patient in needthereof.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Administering” or “administration of” a medicament to a patient (andgrammatical equivalents of this phrase) refers to direct administration,which may be administration to a patient by a medical professional ormay be self-administration, and/or indirect administration, which may bethe act of prescribing a drug. E.g., a physician who instructs a patientto self-administer a medicament or provides a patient with aprescription for a drug is administering the drug to the patient.

The term “affibody” refers to a protein that is derived from the Zdomain of protein A and that been engineered to bind to a specifictarget (see Frejd & Kim, 2017. Exp Mol Med. 49(3): e306).

The term “antibody” refers to a molecule comprising at least oneimmunoglobulin domain that binds to, or is immunologically reactivewith, a particular target. The term includes whole antibodies and anyantigen binding portion or single chains thereof and combinationsthereof; for instance, the term “antibody” in particular includesbivalent antibodies and bivalent bispecific antibodies.

A typical type of antibody comprises at least two heavy chains (“HC”)and two light chains (“LC”) interconnected by disulfide bonds.

Each “heavy chain” comprises a “heavy chain variable domain”(abbreviated herein as “VH”) and a “heavy chain constant domain”(abbreviated herein as “CH”). The heavy chain constant domain typicallycomprises three constants domains, CH1, CH2, and CH3.

Each “light chain” comprises a “light chain variable domain”(abbreviated herein as “VL”) and a “light chain constant domain” (“CL”).The light chain constant domain (CL) can be of the kappa type or of thelambda type. The VH and VL domains can be further subdivided intoregions of hypervariability, termed Complementarity Determining Regions(“CDR”), interspersed with regions that are more conserved, termed“framework regions” (“FW”).

Each VH and VL is composed of three CDRs and four FWs, arranged fromamino-terminus to carboxy-terminus in the following order: FW1, CDR1,FW2, CDR2, FW3, CDR3, FW4. The present disclosure inter alia presents VHand VL sequences as well as the subsequences corresponding to CDR1,CDR2, and CDR3.

The precise amino acid sequence boundaries of a given CDR can bedetermined using any of a number of well-known schemes, including thosedescribed by Kabat et al. (1991), “Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (“Kabat” numbering scheme),Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numberingscheme).

Accordingly, a person skilled in the art would understand that thesequences of FW1, FW2, FW3 and FW4 are equally disclosed. For aparticular VH, FW1 is the subsequence between the N-terminus of the VHand the N-terminus of H-CDR1, FW2 is the subsequence between theC-terminus of H-CDR1 and the N-terminus of H-CDR2, FW3 is thesubsequence between the C-terminus of H-CDR2 and the N-terminus ofH-CDR3, and FW4 is the subsequence between the C-terminus of H-CDR3 andthe C-terminus of the VH. Similarly, for a particular VL, FW1 is thesubsequence between the N-terminus of the VL and the N-terminus ofL-CDR1, FW2 is the subsequence between the C-terminus of L-CDR1 and theN-terminus of L-CDR2. FW3 is the subsequence between the C-terminus ofL-CDR2 and the N-terminus of L-CDR3, and FW4 is the subsequence betweenthe C-terminus of L-CDR3 and the C-terminus of the VL.

The variable domains of the heavy and light chains contain a region thatinteracts with a binding target, and this region interacting with abinding target is also referred to as an “antigen-binding site” or“antigen binding site” herein. The constant domains of the antibodiescan mediate the binding of the antibody to host tissues or factors,including various cells of the immune system (e.g., effector cells) andthe first component (C1q) of the classical complement system. Exemplaryantibodies of the present disclosure include typical antibodies, butalso bivalent fragments and variations thereof such as a F(ab′)2.

As used herein, the term “antibody” encompasses intact polyclonalantibodies, intact monoclonal antibodies, bivalent antibody fragments(such as F(ab′)2), multispecific antibodies such as bispecificantibodies, chimeric antibodies, humanized antibodies, human antibodies,and any other modified immunoglobulin molecule comprising an antigenbinding site.

An antibody can be of any the five major classes (isotypes) ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses thereof(e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on the identity oftheir heavy-chain constant domains referred to as alpha, delta, epsilon,gamma, and mu, respectively. The different classes of immunoglobulinshave different and well known subunit structures and three-dimensionalconfigurations. Antibodies can be naked or conjugated to other moleculessuch as therapeutic agents or diagnostic agents to formimmunoconjugates.

The term “anticalin” refers to a protein that is derived from thelipocalin and that been engineered to bind to a specific target (seeSkerra, 2008. FEBS J. 275(11):2677-83).

The term “antigen-binding fragment” or “Fab” refers to an antibodyfragment comprising one constant and one variable domain of each of theheavy and light chain. A Fab fragment may be obtained by digesting anintact monoclonal antibody with papain.

The term “cancer” refers to a group of diseases, which can be defined asany abnormal benign or malignant new growth of tissue that possesses nophysiological function and arises from uncontrolled usually rapidcellular proliferation and has the potential to invade or spread toother parts of the body.

The term “CD1a” refers to a non-polymorphic MHC Class 1 related cellsurface glycoprotein, expressed in association with β-2-microglobulin.CD1a is expressed by cortical thymocytes, Langerhans cells and byinterdigitating cells. CD1a is also expressed by some malignancies of Tcell lineage and in Langerhans cell histiocytosis. CD1a is expressed oncortical thymocytes, epidermal Langerhans cells, dendritic cells, oncertain T-cell leukemias, and in various other tissues. CD1a isstructurally related to the major histocompatibility complex (MHC)proteins and form heterodimers with β-2-microglobulin. Exemplarysequence and data related to human CD1a has been deposited in theUniProtKB database under ID number P06126.

“CD1a-positive” cancer, including a “CD1a-positive” cancerous disease,is one comprising cells, which have CD1a present at their cell surface.The term “CD1a-positive” also refers to a cancer that producessufficient levels of CD1a at the surface of cells thereof, such that aCAR-comprising cell of the present invention has a therapeutic effect,mediated by the binding of the CAR to CD1a. In some embodiments, theCD1a-positive cancer is cortical T-cell acute lymphoblastic leukemia,and T-cell lymphoblastic lymphoma or Langerhans cell histiocytosis(LCH).

The term “CD1a-targeting moiety” refers to a substance that is able tobind CD1a. Within the context of a CAR, a CD1a-targeting moiety targetsT cells to a CD1a-positive cell, preferably a cancer cell. Within thecontext of a CAR, it is to be understood that the CD1a-targeting moietyis genetically encodable.

The term “chimeric antigen receptor” or “CAR” refers to a syntheticreceptor that targets T cells to a chosen antigen and reprograms T cellfunction, metabolism and persistence (see Riviére & Sadelain, 2017. MolTher. 25(5):1117-1124). Similarly, the term “CART” refers to a T cellthat comprises a CAR.

“Combination therapy”, “in combination with” or “in conjunction with” asused herein denotes any form of concurrent, parallel, simultaneous,sequential or intermittent treatment with at least two distincttreatment modalities (i.e., compounds, components, targeted agents ortherapeutic agents). As such, the terms refer to administration of onetreatment modality before, during, or after administration of the othertreatment modality to the subject. The modalities in combination can beadministered in any order. The therapeutically active modalities areadministered together (e.g., simultaneously in the same or separatecompositions, formulations or unit dosage forms) or separately (e.g., onthe same day or on different days and in any order as according to anappropriate dosing protocol for the separate compositions, formulationsor unit dosage forms) in a manner and dosing regimen prescribed by amedical care taker or according to a regulatory agency. In general, eachtreatment modality will be administered at a dose and/or on a timeschedule determined for that treatment modality. Optionally, three ormore modalities may be used in a combination therapy. Additionally, thecombination therapies provided herein may be used in conjunction withother types of treatment. For example, other anti-cancer treatment maybe selected from the group consisting of chemotherapy, surgery,radiotherapy (radiation) and/or hormone therapy, amongst othertreatments associated with the current standard of care for the subject.

A “complete response” or “complete remission” or “CR” indicates thedisappearance of all target lesions as defined in the RECIST v1.1guideline. This does not always mean the cancer has been cured.

The term “costimulatory signaling domain” refers to a signaling moietythat provides to T cells a signal which, in addition to the primarysignal provided by for instance the CD3 chain of the TCR/CD3 complex,mediates a T cell response, including, but not limited to, activation,proliferation, differentiation, cytokine secretion, and the like. Aco-stimulatory domain can include all or a portion of, but is notlimited to, CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, 1COS,lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, and a ligand that specifically binds with CD83. In someembodiments, the co-stimulatory signaling domain is an intracellularsignaling domain that interacts with other intracellular mediators tomediate a cell response including activation, proliferation,differentiation and cytokine secretion, and the like.

The term “designed ankyrin repeat proteins” or “DARPin” refers to aprotein that is derived from an ankyrin repeat that has been engineeredto bind to a specific target (see Plückthun, 2015. Annu Rev PharmacolToxicol. 55:489-511).

“Disease free survival” (DFS) refers to the length of time during andafter treatment that the patient remains free of disease.

As used herein, the term “effective amount” of an agent, e.g., atherapeutic agent such as a CART, is that amount sufficient to effectbeneficial or desired results, for example, clinical results, and, assuch, an “effective amount” depends upon the context in which it isbeing applied. For example, in the context of administering atherapeutic agent that treats T-ALL, an effective amount can reduce thenumber of cancer cells; reduce the tumor size or burden; inhibit (i.e.,slow to some extent and in a certain embodiment, stop) cancer cellinfiltration into peripheral organs; inhibit (i.e., slow to some extentand in a certain embodiment, stop) tumor metastasis; inhibit, to someextent, tumor growth; relieve to some extent one or more of the symptomsassociated with the cancer; and/or result in a favorable response suchas increased progression-free survival (PFS), disease-free survival(DFS), or overall survival (OS), complete response (CR), partialresponse (PR), or, in some cases, stable disease (SD), a decrease inprogressive disease (PD), a reduced time to progression (TTP) or anycombination thereof. The term “effective amount” can be usedinterchangeably with “effective dose,” “therapeutically effectiveamount,” or “therapeutically effective dose”.

The term “fynomer” refers to a protein that is derived from the SH3domain of human Fyn kinase that has been engineered to bind to aspecific target (see Bertschinger et al., 2007. Protein Eng Des Sel.20(2):57-68).

The terms “individual”, “patient” or “subject” are used interchangeablyin the present application to designate a human being and are not meantto be limiting in any way. The “individual”, “patient” or “subject” canbe of any age, sex and physical condition. The term “patient in needthereof” usually refers to a patient who suffers from a CD1a-positivecancer.

“Infusion” or “infusing” refers to the introduction of a therapeuticagent-containing solution into the body through a vein for therapeuticpurposes. Generally, this is achieved via an intravenous bag.

“Intracellular signaling domain” as used herein refers to all or aportion of one or more domains of a molecule (here the chimeric receptormolecule) that provides for activation of a lymphocyte. Intracellulardomains of such molecules mediate a signal by interacting with cellularmediators to result in proliferation, differentiation, activation andother effector functions. Examples of intracellular signaling domainsfor use in a CAR of the invention include the intracellular sequences ofthe CD3 chain, and/or co-receptors that act in concert to initiatesignal transduction following CAR engagement, as well as any derivativeor variant of these sequences and any synthetic sequence that has thesame functional capability. T cell activation can be said to be mediatedby two distinct classes of cytoplasmic signaling sequence: those thatinitiate antigen-dependent primary activation and provide a T cellreceptor like signal (primary cytoplasmic signaling sequences) and thosethat act in an antigen-independent manner to provide a secondary orco-stimulatory signal (secondary cytoplasmic signaling sequences).Primary cytoplasmic signaling sequences that act in a stimulatory mannermay contain signaling motifs which are known as receptor tyrosine-basedactivation motifs or ITAMs. Examples of ITAM containing primarycytoplasmic signaling sequences include those derived from CD3ζ, FcRγ,CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

The term “monobody” refers to a protein that is derived from afibronectin type III domain that has been engineered to bind to aspecific target (see Koide et al., 2013. J Mol Biol. 415(2):393-405).

The term “nanobody” refers to a protein comprising the soluble singleantigen-binding V-domain of a heavy chain antibody, preferably a camelidheavy chain antibody (see Bannas et al., 2017. Front Immunol. 8:1603).

“Overall Survival” (OS) refers to the time from patient enrollment todeath or censored at the date last known alive. OS includes aprolongation in life expectancy as compared to naive or untreatedindividuals or patients. Overall survival refers to the situationwherein a patient remains alive for a defined period of time, such asone year, five years, etc., e.g., from the time of diagnosis ortreatment.

A “partial response” or “PR” refers to at least a 30% decrease in thesum of diameters of target lesions, taking as reference the baseline sumdiameter, in response to treatment, as defined in the RECIST v1.1guideline.

The term “peptide aptamer” refers to a short, 5-20 amino acid residuesequence that can bind to a specific target. Peptide aptamers aretypically inserted within a loop region of a stable protein scaffold(see Reverdatto et al., 2015. Curr Top Med Chem. 15 (12): 1082-101).

As used herein, “pharmaceutically acceptable carrier” or“pharmaceutically acceptable diluent” means any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed and, without limiting the scope of the presentinvention, include: additional buffering agents; preservatives;co-solvents; antioxidants, including ascorbic acid and methionine;chelating agents such as EDTA; metal complexes (e.g., Zn-proteincomplexes); biodegradable polymers, such as polyesters; salt-formingcounterions, such as sodium, polyhydric sugar alcohols; amino acids,such as alanine, glycine, glutamine, asparagine, histidine, arginine,lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, andthreonine; organic sugars or sugar alcohols, such as lactitol,stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose,myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g.,inositol), polyethylene glycol; sulfur containing reducing agents, suchas urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol,[alpha]-monothioglycerol, and sodium thio sulfate; low molecular weightproteins, such as human serum albumin, bovine serum albumin, gelatin, orother immunoglobulins; and hydrophilic polymers, such aspolyvinylpyrrolidone. Other pharmaceutically acceptable carriers,excipients, or stabilizers, such as those described in Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) may also beincluded in a pharmaceutical composition described herein, provided thatthey do not adversely affect the desired characteristics of thepharmaceutical composition.

“Progressive disease” or “disease that has progressed” refers to theappearance of one more new lesions or tumors and/or the unequivocalprogression of existing non-target lesions as defined in the RECIST v1.1guideline. Progressive disease or disease that has progressed can alsorefer to a tumor growth of more than 20 percent since treatment began,either due to an increase in mass or in spread of the tumor.

“Progression free survival” (PFS) refers to the time from enrollment todisease progression or death. PFS is generally measured using theKaplan-Meier method and Response Evaluation Criteria in Solid Tumors(RECIST) 1.1 standards. Generally, progression free survival refers tothe situation wherein a patient remains alive, without the cancergetting worse.

The term “RECIST” means Response Evaluation Criteria in Solid Tumours.RECIST guideline, criteria, or standard, describes a standard approachto solid tumor measurement and definitions for objective assessment ofchange in tumor size for use in adult and pediatric cancer clinicaltrials. RECIST v1.1 means version 1.1 of the revised RECIST guidelineand it is published in European Journal of Cancers 45 (2009) 228-247.

The term “repebody” refers to a protein that is derived from aleucine-rich repeat module and that been engineered to bind to aspecific target (see Lee et al., 2012. PNAS. 109(9): 3299-3304).

The term “respond favorably” generally refers to causing a beneficialstate in a subject. With respect to cancer treatment, the term refers toproviding a therapeutic effect on the subject. Positive therapeuticeffects in cancer can be measured in a number of ways (See, Weber, 2009.J Nucl Med. 50 Suppl 1:1S-10S). For example, tumor growth inhibition,molecular marker expression, serum marker expression, and molecularimaging techniques can all be used to assess therapeutic efficacy of ananti-cancer therapeutic. With respect to tumor growth inhibition,according to NCI standards, a T/C≤42% is the minimum level of anti-tumoractivity. A T/C≤10% is considered a high anti-tumor activity level, withT/C (%)=Median tumor volume of the treated/Median tumor volume of thecontrol×100. A favorable response can be assessed, for example, byincreased progression-free survival (PFS), disease-free survival (DFS),or overall survival (OS), complete response (CR), partial response (PR),or, in some cases, stable disease (SD), a decrease in progressivedisease (PD), a reduced time to progression (TTP) or any combinationthereof.

The term “sequence identity” refers to a percentage value obtained whentwo sequences are compared using a pairwise sequence alignment tool. Inthe present case, the sequence identity is obtained using the globalalignment tool “EMBOSS Needle” using the default settings (Rice et al.,2000. Trends Genet. 16(6):276-7; Li et al., 2015. Nucleic Acids Res.43(W1):W580-4). The global alignment tool is available at:https://www.ebi.ac.uk/Tools/psa/.

The term “single-chain antigen-binding fragment” or “scFab” refers to afusion protein comprising one variable and one constant domain of thelight chain of an antibody attached to one variable and one constantdomain of the heavy chain of an antibody, wherein the heavy and lightchains are linked together through a short peptide.

The term “single-chain variable fragment” or “scFv” refers to a fusionprotein comprising the variable domains of the heavy chain and lightchain of an antibody linked to one another with a peptide linker. Theterm also includes a disulfide stabilized Fv (dsFv). Methods ofstabilizing scFvs with disulfide bonds are disclosed in Reiter et al.,1996. Nat Biotechnol. 14(10):1239-45.

“Stable disease” refers to disease without progression or relapse asdefined in the RECIST v1.1 guideline. In stable disease there is neithersufficient tumor shrinkage to qualify for partial response, norsufficient tumor increase to qualify as progressive disease.

“Time to Tumor Progression” (TTP) is defined as the time from enrollmentto disease progression. TTP is generally measured using the RECIST v1.1criteria.

The terms “treatment” and “therapy”, as used in the present application,refer to a set of hygienic, pharmacological, surgical and/or physicalmeans used with the intent to cure and/or alleviate a disease and/orsymptoms with the goal of remediating the health problem. The terms“treatment” and “therapy” include preventive and curative methods, sinceboth are directed to the maintenance and/or reestablishment of thehealth of an individual or animal. Regardless of the origin of thesymptoms, disease and disability, the administration of a suitablemedicament to alleviate and/or cure a health problem should beinterpreted as a form of treatment or therapy within the context of thisapplication.

Chimeric Antigen Receptor

In one aspect, the present invention provides a chimeric antigenreceptor (CAR) comprising an extracellular domain comprising a CD1atargeting-moiety, a transmembrane domain, and an intracellular signalingdomain.

CD1a Targeting-Moiety

In some embodiments, the CD1a-targeting moiety is an antibody,anticalin, repebody, monobody, scFv, Fab, scFab, affibody, fynomer,DARPin, nanobody, or peptide aptamer that specifically binds to CD1a.

Binding molecules that bind specifically to CD1a may be very useful inthe diagnosis and treatment of the disorders mentioned above. Severalmurine monoclonal antibodies against CD1a are known in the field (Kelly(1994), Amiot et al. (1986), Fume et al. (1992)). However, murineantibodies are limited for in vivo use due to issues associated with theadministration of murine antibodies to humans, such as short serumhalf-life, the inability to trigger certain human effector functions andthe generation of an undesired immune response against the murineantibody (Van Kroonenburgh and Pauwels (1988)). New human antibodieshave been developed (Bechan (2012), and Gitanjali (2005) in recent yearsovercoming these previously mentioned drawbacks. Besides NA1/34.HLK,other hybridomas are commercially available, e.g. OKT6 (IgG1 isotype),from SIGMA ALDRICH.

Please refer to:

-   Amiot M., Bernard A., Raynal B., Knapp W., Deschildre C. and    Boumsell L. (1986), J. Immunol. 136:1752-1757.-   Fume M., Nindl M., Kawabe K., Nakamura K., Ishibashi Y. and    Sagawa K. (1992), J. Am. Acad. Dermatol. 27:419-42-   Kelly K. M., Beverly P. C., Chu A. C., Davenport V., Gordon I.,    Smith M. and Pritchard J. (1994), J. Pediatr. 125:717-722-   Van Kroonenburgh M. J. and Pauwels E. K. (1988), Nucl. Med. Commun.    9:919-930.-   Gitanjali Bechan, David W. Lee, R. Maarten Egeler and Robert J.    Arceci Blood 2005 106:4815-   Bechan, G. I., Lee, D. W., Zajonc, D. M., Heckel, D. , Xian, R. ,    Throsby, M. , Meijer, M. , Germeraad, W. T., Kruisbeek, A. M.,    Maarten Egeler, R. and Arceci, R. J. (2012), Br J Haematol, 159:    299-310.

Phage display and combinatorial methods for generating antibodies areknown in the art (as described in, e.g., Ladner et al. U.S. Pat. No.5,223,409; Kang et al. International Publication No. WO 92/18619; Doweret al. International Publication No. WO 91/17271; Winter et al.International Publication WO 92/20791; Markland et al. InternationalPublication No. WO 92/15679; Breitling et al. International PublicationWO 93/01288; McCafferty et al. International Publication No. WO92/01047; Garrard et al. International Publication No. WO 92/09690;Ladner et al. International Publication No. WO 90/02809; Fuchs et al.(1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum AntibodHybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffthset al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) PNAS 88:7978-7982, the contents of all of which areincorporated by reference herein).

Further, methods of generating and selecting non-immunglobulin scaffoldsthat bind to a particular target are known in the art (see, for example,Skrlec, et al., 2015. Trends Biotechnol. 33(7):408-18).

In some embodiments, the CD1a-targeting moiety is an antibody, scFv,Fab, or scFab comprising a VL domain and VH domain, wherein said VLdomain comprises LCDR1, LCDR2 and LCDR3 polypeptides and said VH domaincomprises HCDR1, HCDR2 and HCDR3 polypeptides, and LCDR1 consists of[QDINKY] (SEQ ID NO: 1), LCDR2 consists of [YTS], LCDR3 consists of[LHYDNLPWT] (SEQ ID NO: 3), HCDR1 consists of [GYAFSTYT] (SEQ ID NO: 4),HCDR2 consists of [INPNSAST] (SEQ ID NO: 5), and HCDR3 consists of[ARGFYTMDY] (SEQ ID NO: 6).

In some embodiments, the CD1a-targeting moiety is a scFv comprising a VLdomain and VH domain, wherein said VL domain comprises LCDR1, LCDR2 andLCDR3 polypeptides and said VH domain comprises HCDR1, HCDR2 and HCDR3polypeptides, and LCDR1 consists of [QDINKY] (SEQ ID NO: 1), LCDR2consists of [YTS], LCDR3 consists of [LHYDNLPWT] (SEQ ID NO: 3), HCDR1consists of [GYAFSTYT] (SEQ ID NO: 4), HCDR2 consists of [INPNSAST] (SEQID NO: 5), and HCDR3 consists of [ARGFYTMDY] (SEQ ID NO: 6).

In some embodiments, the CD1a-targeting moiety is an antibody, scFv,Fab, or scFab comprising a VL domain and VH domain, wherein the VLdomain consists of SEQ ID NO: 7 and the VH domain consists of SEQ ID NO:8.

In some embodiments, the CD1a-targeting moiety is a scFv comprising a VLdomain and VH domain, wherein the VL domain consists of SEQ ID NO: 7 andthe VH domain consists of SEQ ID NO: 8.

VL domain (SEQ ID NO: 7)[RDIQMTQSPSSLSASLGGKVTITCQASQDINKYIAWYQFKPGKGPRLLIHYTSTLQPAIPSRFSGSGSGREYSFSISNLEPEDIATYYCLHYDNLPWTFGG GTKLEIKRA] VH domain(SEQ ID NO: 8) [QVQLQQSGAELARPGASVKMSCKASGYAFSTYTMHWVKQRPRQGLEWIGYINPNSASTSYNENFKDKATLTADKSSNTAYMHLSSLTSEDSAVYYCARGFY TMDYWGQGTSVTVSS]

In some embodiments, the CD1a-targeting moiety is a scFv comprising orconsisting of SEQ ID NO: 9.

scFv derived from clone NA1/34.HLK (SEQ ID NO: 9)[QVQLQQSGAELARPGASVKMSCKASGYAFSTYTMHWVKQRPRQGLEWIGYINPNSASTSYNENFKDKATLTADKSSNTAYMHLSSLTSEDSAVYYCARGFYTMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSRDIQMTQSPSSLSASLGGKVTITCQASQDINKYIAWYQFKPGKGPRLLIHYTSTLQPAIPSRFSGSGSGREYSFSISNLEPEDIATYYCLHYDNLPWTFGGGTKLEIKRA]

Transmembrane Domain

The transmembrane domain may be derived either from a natural or asynthetic source. When the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsmay comprise at least the transmembrane region(s) of the α-, β- orζ-chain of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64,CD80, CD86, CD134, CD137, or CD154.

A transmembrane domain may be synthetic or a variant of a naturallyoccurring transmembrane domain. In some embodiments, synthetic orvariant transmembrane domains comprise predominantly hydrophobicresidues such as leucine and valine.

In some embodiments, the transmembrane domain comprises thetransmembrane domain of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22,CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a variant thereof,wherein the variant thereof has a 95% sequence identity.

In some embodiments, the transmembrane domain comprises thetransmembrane domain of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22,CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a variant thereof,wherein the variant thereof has a 98% sequence identity.

In some embodiments, the transmembrane domain comprises thetransmembrane domain of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22,CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.

In some embodiments, the transmembrane domain comprises thetransmembrane domain of CD8 or a variant thereof, wherein the variantthereof has a 95% sequence identity.

In some embodiments, the transmembrane domain comprises thetransmembrane domain of CD8 or a variant thereof, wherein the variantthereof has a 98% sequence identity.

In some embodiments, the transmembrane domain comprises thetransmembrane domain of CD8.

In some embodiments, the transmembrane domain comprises SEQ ID NO: 10 ora sequence that has 95% sequence identity to SEQ ID NO: 10.

In some embodiments, the transmembrane domain comprises SEQ ID NO: 10 ora sequence that has 98% sequence identity to SEQ ID NO: 10.

In some embodiments, the transmembrane domain comprises SEQ ID NO: 10.In some embodiments, the transmembrane domain consists of SEQ ID NO: 10.

Transmembrane domain derived from CD8 (SEQ ID NO: 10)[TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYC]

Intracellular Signaling Domain

The intracellular signaling domain provides for the activation of atleast one function of the cell expressing the CAR upon binding to theligand expressed on tumor cells. In some embodiments, the intracellularsignaling domain contains one or more intracellular signaling domains.In some embodiments, the intracellular signaling domain is a portion ofand/or a variant of an intracellular signaling domain that provides foractivation of at least one function of the CAR-comprising cell.

In some embodiments, the intracellular signaling domain comprises theintracellular domain of CD3ζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a,CD79b, CD66b, or a variant thereof, wherein the variant thereof has a95% sequence identity.

In some embodiments, the intracellular signaling domain comprises theintracellular domain of CD3ζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a,CD79b, CD66b, or a variant thereof, wherein the variant thereof has a98% sequence identity.

In some embodiments, the intracellular signaling domain comprises theintracellular domain of CD3ζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a,CD79b or CD66b.

In some embodiments, the intracellular signaling domain comprises theintracellular domain of CD3 or a variant thereof, wherein the variantthereof has a 95% sequence identity.

In some embodiments, the intracellular signaling domain comprises theintracellular domain of CD3 or a variant thereof, wherein the variantthereof has a 98% sequence identity.

In some embodiments, the intracellular signaling domain comprises theintracellular domain of CD3.

In some embodiments, the intracellular signaling domain comprises SEQ IDNO: 11 or a sequence that has 95% sequence identity to SEQ ID NO: 11.

In some embodiments, the intracellular signaling domain comprises SEQ IDNO: 11 or a sequence that has 98% sequence identity to SEQ ID NO: 11.

In some embodiments, the intracellular signaling domain comprises SEQ IDNO: 11 or a sequence that has 99% sequence identity to SEQ ID NO: 11.

In some embodiments, the intracellular signaling domain comprises SEQ IDNO: 11. In some embodiments, the intracellular signaling domain consistsof SEQ ID NO: 11.

Intracellular signaling domain derived from CD3ζ (SEQ ID NO: 11)[RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR]

Costimulatory Signaling Domain

In some embodiments, the CAR may further comprise a costimulatorysignaling domain. In some embodiments, the costimulatory signalingdomain comprises the intracellular domain of CD27, CD28, CD137, CD134,CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7,LIGHT, NKG2C, CD276 or a variant thereof, wherein the variant thereofhas a 95% sequence identity.

In some embodiments, the costimulatory signaling domain comprises theintracellular domain of CD27, CD28, CD137, CD134, CD30, CD40, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, CD276 ora variant thereof, wherein the variant thereof has a 98% sequenceidentity.

In some embodiments, the costimulatory signaling domain comprises theintracellular domain of CD27, CD28, CD137, CD134, CD30, CD40, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or CD276.

In some embodiments, the costimulatory signaling domain comprises theintracellular domain of CD137 or a variant thereof, wherein the variantthereof has a 95% sequence identity.

In some embodiments, the costimulatory signaling domain comprises theintracellular domain of CD137 or a variant thereof, wherein the variantthereof has a 98% sequence identity.

In some embodiments, the costimulatory signaling domain comprises theintracellular domain of CD137.

In some embodiments, the costimulatory signaling domain comprises SEQ IDNO: 12 or a sequence that has 95% sequence identity to SEQ ID NO: 12.

In some embodiments, the costimulatory signaling domain comprises SEQ IDNO: 12 or a sequence that has 98% sequence identity to SEQ ID NO: 12.

In some embodiments, the costimulatory signaling domain comprises SEQ IDNO: 12 or a sequence that has 99% sequence identity to SEQ ID NO: 12.

In some embodiments, the costimulatory signaling domain comprises SEQ IDNO: 12. In some embodiments, the costimulatory signaling domain consistsof SEQ ID NO: 12.

Costimulatory signaling domain derived from CD137 (SEQ ID NO: 12)[KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL]

Full Sequence CARs According to the Present Invention

In some embodiments, the CAR comprises:

(i) a scFv comprising a VL domain and VH domain, wherein said VL domaincomprises LCDR1, LCDR2 and LCDR3 polypeptides and said VH domaincomprises HCDR1, HCDR2 and HCDR3 polypeptides, and LCDR1 consists of[QDINKY] (SEQ ID NO: 1), LCDR2 consists of [YTS], LCDR3 consists of[LHYDNLPWT] (SEQ ID NO: 3), HCDR1 consists of [GYAFSTYT] (SEQ ID NO: 4),HCDR2 consists of [INPNSAST] (SEQ ID NO: 5), and HCDR3 consists of[ARGFYTMDY] (SEQ ID NO: 6);(ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence thathas 95% sequence identity to SEQ ID NO: 10;(iii) an intracellular signaling domain comprising SEQ ID NO: 11 or asequence that has 95% sequence identity to SEQ ID NO: 11; and(iv) a costimulatory signaling domain comprising SEQ ID NO: 12 or asequence that has 95% sequence identity to SEQ ID NO: 12.

In some embodiments, the CAR comprises:

(i) a scFv comprising a VL domain and VH domain, wherein said VL domaincomprises LCDR1, LCDR2 and LCDR3 polypeptides and said VH domaincomprises HCDR1, HCDR2 and HCDR3 polypeptides, and LCDR1 consists of[QDINKY] (SEQ ID NO: 1), LCDR2 consists of [YTS], LCDR3 consists of[LHYDNLPWT] (SEQ ID NO: 3), HCDR1 consists of [GYAFSTYT] (SEQ ID NO: 4),HCDR2 consists of [INPNSAST] (SEQ ID NO: 5), and HCDR3 consists of[ARGFYTMDY] (SEQ ID NO: 6);(ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence thathas 98% sequence identity to SEQ ID NO: 10;(iii) an intracellular signaling domain comprising SEQ ID NO: 11 or asequence that has 98% sequence identity to SEQ ID NO: 11; and(iv) a costimulatory signaling domain comprising SEQ ID NO: 12 or asequence that has 98% sequence identity to SEQ ID NO: 12.

In some embodiments, the CAR comprises:

(i) a scFv comprising a VL domain and VH domain, wherein said VL domaincomprises LCDR1, LCDR2 and LCDR3 polypeptides and said VH domaincomprises HCDR1, HCDR2 and HCDR3 polypeptides, and LCDR1 consists of[QDINKY] (SEQ ID NO: 1), LCDR2 consists of [YTS], LCDR3 consists of[LHYDNLPWT] (SEQ ID NO: 3), HCDR1 consists of [GYAFSTYT] (SEQ ID NO: 4),HCDR2 consists of [INPNSAST] (SEQ ID NO: 5), and HCDR3 consists of[ARGFYTMDY] (SEQ ID NO: 6);(ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence thathas 98% sequence identity to SEQ ID NO: 10;(iii) an intracellular signaling domain comprising SEQ ID NO: 11 or asequence that has 99% sequence identity to SEQ ID NO: 11; and(iv) a costimulatory signaling domain comprising SEQ ID NO: 12 or asequence that has 99% sequence identity to SEQ ID NO: 12.

In some embodiments, the CAR comprises:

(i) a scFv comprising a VL domain and VH domain, wherein said VL domaincomprises LCDR1, LCDR2 and LCDR3 polypeptides and said VH domaincomprises HCDR1, HCDR2 and HCDR3 polypeptides, and LCDR1 consists of[QDINKY] (SEQ ID NO: 1), LCDR2 consists of [YTS], LCDR3 consists of[LHYDNLPWT] (SEQ ID NO: 3), HCDR1 consists of [GYAFSTYT] (SEQ ID NO: 4),HCDR2 consists of [INPNSAST] (SEQ ID NO: 5), and HCDR3 consists of[ARGFYTMDY] (SEQ ID NO: 6);(ii) a transmembrane domain comprising SEQ ID NO: 10;(iii) an intracellular signaling domain comprising SEQ ID NO: 11; and(iv) a costimulatory signaling domain comprising SEQ ID NO: 12.

In some embodiments, the CAR comprises:

(i) a scFv comprising a VL domain and VH domain, wherein said VL domaincomprises LCDR1, LCDR2 and LCDR3 polypeptides and said VH domaincomprises HCDR1, HCDR2 and HCDR3 polypeptides, and LCDR1 consists of[QDINKY] (SEQ ID NO: 1), LCDR2 consists of [YTS], LCDR3 consists of[LHYDNLPWT] (SEQ ID NO: 3), HCDR1 consists of [GYAFSTYT] (SEQ ID NO: 4),HCDR2 consists of [INPNSAST] (SEQ ID NO: 5), and HCDR3 consists of[ARGFYTMDY] (SEQ ID NO: 6);(ii) a transmembrane domain consisting of SEQ ID NO: 10;(iii) an intracellular signaling domain consisting of SEQ ID NO: 11; and(iv) a costimulatory signaling domain consisting of SEQ ID NO: 12.

In some embodiments, the CAR comprises:

(i) a scFv comprising a VL domain and VH domain, wherein the VL domainconsists of SEQ ID NO: 7 and the VH domain consists of SEQ ID NO: 8;(ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence thathas 95% sequence identity to SEQ ID NO: 10;(iii) an intracellular signaling domain comprising SEQ ID NO: 11 or asequence that has 95% sequence identity to SEQ ID NO: 11; and(iv) a costimulatory signaling domain comprising SEQ ID NO: 12 or asequence that has 95% sequence identity to SEQ ID NO: 12.

In some embodiments, the CAR comprises:

(i) a scFv comprising a VL domain and VH domain, wherein the VL domainconsists of SEQ ID NO: 7 and the VH domain consists of SEQ ID NO: 8;(ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence thathas 98% sequence identity to SEQ ID NO: 10;(iii) an intracellular signaling domain comprising SEQ ID NO: 11 or asequence that has 98% sequence identity to SEQ ID NO: 11; and(iv) a costimulatory signaling domain comprising SEQ ID NO: 12 or asequence that has 98% sequence identity to SEQ ID NO: 12.

In some embodiments, the CAR comprises:

(i) a scFv comprising a VL domain and VH domain, wherein the VL domainconsists of SEQ ID NO: 7 and the VH domain consists of SEQ ID NO: 8;(ii) a transmembrane domain comprising SEQ ID NO: 10 or a sequence thathas 98% sequence identity to SEQ ID NO: 10;(iii) an intracellular signaling domain comprising SEQ ID NO: 11 or asequence that has 99% sequence identity to SEQ ID NO: 11; and(iv) a costimulatory signaling domain comprising SEQ ID NO: 12 or asequence that has 99% sequence identity to SEQ ID NO: 12.

In some embodiments, the CAR comprises:

(i) a scFv comprising a VL domain and VH domain, wherein the VL domainconsists of SEQ ID NO: 7 and the VH domain consists of SEQ ID NO: 8;(ii) a transmembrane domain comprising SEQ ID NO: 10;(iii) an intracellular signaling domain comprising SEQ ID NO: 11; and(iv) a costimulatory signaling domain comprising SEQ ID NO: 12.

In some embodiments, the CAR comprises:

(i) a scFv comprising a VL domain and VH domain, wherein the VL domainconsists of SEQ ID NO: 7 and the VH domain consists of SEQ ID NO: 8;(ii) a transmembrane domain consisting of SEQ ID NO: 10;(iii) an intracellular signaling domain consisting of SEQ ID NO: 11; and(iv) a costimulatory signaling domain consisting of SEQ ID NO: 12.

In some embodiments, the CAR comprises or consists of SEQ ID NO: 2 or asequence that has 95% sequence identity with SEQ ID NO: 2. In someembodiments, the CAR comprises or consists of SEQ ID NO: 2 or a sequencethat has 98% sequence identity with SEQ ID NO: 2. In some embodiments,the CAR comprises or consists of SEQ ID NO: 2 or a sequence that has 99%sequence identity with SEQ ID NO: 2. In some embodiments, the CARcomprises or consists of SEQ ID NO: 2.

Full sequence of the CAR (SEQ ID NO: 2)[MALPVTGLLLSLGLLLHAARPTGQVQLQQSGAELARPGASVKMSCKASGYAFSTYTMHWVKQRPRQGLEWIGYINPNSASTSYNENFKDKATLTADKSSNTAYMHLSSLTSEDSAVYYCARGEYTMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSRDIQMTQSPSSLSASLGGKVTITCQASQDINKYIAWYQFKPGKGPRLLIHYTSTLQPAIPSRFSGSGSGREYSFSISNLEPEDIATYYCLHYDNLPWTFGGGTKLEIKRATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR]

Nucleic Acid

In one aspect, the present invention provides a nucleic acid encodingany one of the CARS of the present invention, including any one of theCARS disclosed above. The nucleic acid sequence that encodes thechimeric receptor links together a number of modular components that canbe excised and replaced with other components in order to customize thechimeric receptor for efficient T cell activation and recognition ofCD1a.

In some embodiments, the nucleic acid is suitable for transducing ortransforming a cell. In some embodiments, the nucleic acid is suitablefor transducing or transforming a T cell for use in adoptiveimmunotherapy.

In some embodiments, the nucleic acid is codon optimized for expressionin mammalian cells. Codon optimization methods are known in the art(see, for example, Parret et al., 2016. Curr Opin Struct Biol. 39:155-162).

The nucleic acid of the present invention may be comprised in aγ-retroviral or lentiviral vector which can be used to transduce ortransform a T cell (see Riviére & Sadelain, 2017. Mol Ther.25(5):1117-1124). The nucleic acid may also be inserted into a cellthrough the use of DNA transposons, RNA transfection or genome editingtechniques such as TALEN, ZFN and CRISPR/Cas9 (see Riviére & Sadelain,2017. Mol Ther. 25(5):1117-1124).

Cells

In one aspect, the present invention provides a cell comprising thenucleic acid of the present invention and/or the CAR of the presentinvention. In some embodiments, the cell is a T-cell (referred to as aCART).

In some embodiments, the cell is a naïve T cell, memory stem T cell orcentral memory T cell. It is currently thought that these cells arebetter suited for adaptive immunotherapy (see Rivière & Sadelain, 2017.Mol Ther. 25(5):1117-1124).

In some embodiments, the cell is an autologous T cell. The term“autologous cell” refers to a cell obtained from the same patient thatis to be treated using any one of the methods of the present invention.It is noted that flow cytometric analysis of peripheral blood obtainedfrom 40 patients with active T-cell acute lymphoblastic leukemiarevealed the presence of normal CD3+CD1a− T-cells in all the patients.Thus, it is entirely possible to treat a patient using an autologous Tcell comprising the nucleic acid and/or CAR of the present invention.

In some embodiments, the cell is an allo-tolerant T cell. The term“allo-tolerant cell” refers to a cell that has been engineered todecrease the risk of a Graft-versus-host disease response. In someembodiments, this is achieved by genomic editing-mediated deletion ofTCR and/or β2-microglobulin^(15,19). Allo-tolerant cells are known inthe art (see section of allogeneic T cells in Riviére & Sadelain, 2017.Mol Ther. 25(5):1117-1124).

In some embodiments, the T cell is a CD3-positive and CD1a-negative Tcell.

In some embodiments, the cell is a lymphoid precursor, embryonic stemcell or an induced pluripotent stem cell with the capacity todifferentiate into a mature T cell (see Riviére & Sadelain, 2017. MolTher. 25(5):1117-1124).

Pharmaceutical Composition

In one aspect, the present invention provides a pharmaceuticalcomposition comprising a plurality of cells of the present invention anda pharmaceutically acceptable carrier or diluent.

A pharmaceutical composition as described herein may also contain othersubstances. These substances include, but are not limited to,cryoprotectants, surfactants, anti-oxidants, and stabilizing agents. Theterm “cryoprotectant” as used herein, includes agents which providestability to the CARTs against freezing-induced stresses. Non-limitingexamples of cryoprotectants include sugars, such as sucrose, glucose,trehalose, mannitol, mannose, and lactose; polymers, such as dextran,hydroxyethyl starch and polyethylene glycol; surfactants, such aspolysorbates (e.g., PS-20 or PS-80); and amino acids, such as glycine,arginine, leucine, and serine. A cryoprotectant exhibiting low toxicityin biological systems is generally used.

In some embodiments, the cells are formulated by first harvesting themfrom their culture medium, and then washing and concentrating the cellsin a medium and container system suitable for administration (a“pharmaceutically acceptable” carrier) in a therapeutically effectiveamount. Suitable infusion medium can be any isotonic medium formulation,typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter),but also 5% dextrose in water or Ringer's lactate can be utilized. Theinfusion medium can be supplemented with human serum albumin, fetalbovine serum or other human serum components.

In one aspect, the present invention provides a cell according to thepresent invention or a pharmaceutical composition according to thepresent invention for use as a medicament.

Methods of Treatment

In one aspect, the present invention provides a method of treating aCD1a-positive cancer comprising administering the cell of the presentinvention or the pharmaceutical composition of the present invention toa patient in need thereof.

In some embodiments, the patient is administered a therapeuticallyeffective amount of cells. In some embodiments, the patient isadministered at least 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹ or 10¹⁰cells. The number of cells will depend upon the ultimate use for whichthe composition is intended as will the type of cells included therein.For example, if cells that are specific for a particular antigen aredesired, then the population will contain greater than 70%, generallygreater than 80%, 85% and 90-95% of such cells. For uses providedherein, the cells are generally in a volume of a liter or less, can be500 ml or less, even 250 ml or less, or 100 ml or less. The clinicallyrelevant number of cells can be apportioned into multiple infusions thatcumulatively equal or exceed 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹ or10¹⁰ cells.

In some embodiments, the cell or pharmaceutical composition isadministered intravenously, intraperitoneally, into the bone marrow,into the lymph node, and/or into cerebrospinal fluid.

In some embodiments, the method comprises a combination therapy. In someembodiments, the method comprises further administering an immunecheckpoint inhibitor (see Lim & June, 2017. Cell. 168(4):724-740). In afurther embodiment, the method comprises further administering an immunecheckpoint inhibitor and/or an IAP inhibitor (see WO 2016/054555).

In some embodiments, the cell or pharmaceutical composition as describedherein is administered in combination with chemotherapeutic agentsand/or immunosuppressants. In an embodiment, a patient is first treatedwith a chemotherapeutic agent that inhibits or destroys other immunecells followed by the cell or pharmaceutical composition describedherein. In some cases, chemotherapy may be avoided entirely.

In some embodiments, the CD1a-positive cancer is cortical T-cell acutelymphoblastic leukemia or Langerhans cell histiocytosis. In someembodiments, the CD1a-positive cancer is cortical T-cell acutelymphoblastic leukemia. In some embodiments, the CD1a-positive cancer isrelapsed/refractory cortical T-cell acute lymphoblastic leukemia.

In general, the relapse of leukemia can manifest several months or yearsafter the initial remission; however, most relapses occur within twoyears after the initial treatment. Refractoriness is a term that impliesthat the patient has no longer responded to at least one therapystrategy after a relapse.

There is a broad consensus in first-line trials for ALL, specifically inadults that a relapse is defined as “detection of more than 5% of blastcells in the bone marrow after a previous achievement of completeremission (CR) or unequivocal demonstration of extramedullary leukemiaparticipation” (see Gökbuget (2017)). The European Working Group onAdult ALL (EWALL) has documented this statement in a consensusrecommendation, (see Dohner (2010)) with the additional explanation that“in the case of 5 to 20% of cell blasts at some stage during theintensive treatment phase and/or during regeneration, the evaluation ofthe bone marrow should be repeated one week later to distinguish amongbone marrow relapse and regeneration phenomenon”. The cited definitionis based on international recommendations for outcome parameters inacute myeloid leukemia (see Cheson (2003) and Chantepie (213)); that hasbeen extrapolated to several subtypes of ALL, as in the case of T-ALL.

More recently, some trials did not even define the concept of relapse.Therefore, studies with chimeric antigen receptor (CAR) T cells includedpatients with “measurable disease” and also included patients withhaematological relapse (no additional specification) or minimal residualdisease (MRE) (see Lee (2015) and Maude (2014) and Gokbuget (2017)).Please refer to:

-   Dohner H, Estey E H, Amadori S, et al, Diagnosis and management of    acute myeloid leukemia in adults: recommendations from an    international expert panel, on behalf of the European Leukemia Net.    Blood 2010; 115:453-74.-   Cheson B D, Bennett J M, Kopecky K J, et al. Revised recommendations    of the International Working Group for Diagnosis, Standardization of    Response Criteria, Treatment Outcomes, and Reporting Standards for    Therapeutic Trials in Acute Myeloid Leukemia. J Clin Oncol 2003;    21:4642-9.-   Chantepie S P, Cornet E, Salaun V, Reman O. Hematogones: an    overview. Leuk Res 2013; 37:1404-11.-   13. Maude S L, Frey N, Shaw P A, et al. Chimeric antigen receptor T    cells for sustained remissions in leukemia. N Engl J Med 2014;    371:1507-17.-   Gökbuget N, Dombret H, Bassan R, Wadleigh M, Doubek M, Ribera J.    Inclusion and response criteria for clinical trials in    relapsed/refractory acute lymphoblastic leukemia and usefulness of    historical control trials. Haematologica. 2017; 102(3): e118-e119.

In some embodiments, the patient to be treated with the method of thepresent invention is in complete or near-complete remission aftertreatment with another therapy. It may be preferable desirable todecrease the tumor burden before using the methods of the presentinvention because since there are several alternative effector T-cellsin cases of patients with highly active relapsed/refractory corticalT-cell acute lymphoblastic leukemia. In some embodiments, the patient tobe treated with the method of the present invention has previously beentreated with another therapy which resulted in a partial response,complete response, stable disease, decrease in progressive disease,reduced time to tumor progression or any combination thereof.

EXAMPLES Materials and Methods

CD1a-Specific scFv Generation and CAR Design

The CD1a-specific single-chain variable fragment (scFv) derived from theNA1/34.HLK clone of CD1a-specific antibody was obtained using commercialsynthesis (Sigma-Aldrich) with the mouse IgG Library Primer Set(Progen), and was cloned into a pCCL lentiviral-based second-generationCAR backbone containing a human CD8 transmembrane (TM) domain, humanCD137 and CD3 endodomains, and a T2A-GFP cassette. Identical lentiviralvectors expressing either GFP alone (mock vector) or CD22 CAR backbonewere used as controls (FIGS. 1D & 8A).

CAR-Expressing Lentiviral Production, T-Cell Transduction, Activationand Expansion

CAR-expressing viral particles pseudotyped with VSV-G were generated in293T cells using a standard polyethylenimine transfection protocol, andwere concentrated by ultracentrifugation as described elsewhere²⁷. Viraltiters were consistently in the range of 10⁸ TU/mL. Peripheral bloodmononuclear cells (PBMCs) were isolated from buffy coats from healthyvolunteers by Ficoll-Hypaque gradient centrifugation. Buffy coats wereobtained from the Barcelona Blood and Tissue Bank (BST) upon IRBapproval (HCB/2018/0030). T-cells were activated by plate-bound anti-CD3(OKT3) and anti-CD28 antibodies (BD Biosciences) for 2 days and werethen transduced with CAR-expressing lentivirus (MOI=10) in the presenceof interleukin-7 (IL-7) and IL-15 (10 ng/mL, Mitenyi Biotec)^(16,18).The cell surface expression of CD1aCAR was traced byfluorescence-activated cell sorting (FACS) coexpression of GFP and wasconfirmed using an AffiniPure F(ab′)2 Fragment Goat Anti-Mouse IgG (H+L)(Jackson ImmunoResearch). Proper activation of CAR-transduced T cellswas demonstrated by staining for CD25 and CD69 after 2-day expansion.

Immunophenotyping of Healthy CD34+ Progenitors, T-Cells and PrimaryT-ALL Samples

The expression of CD1a antigen in CD34+ stem/progenitor cells (HSPCs),CD34+CD7+ thymic T-cell progenitors and CD3+ T-cells was prospectivelyanalyzed in fresh human thymus, fetal liver and bone marrow (BM), cordblood and adult BM and peripheral blood (PB) (n=3). Fetal tissue wascollected as previously described 28,29 from developing embryos abortedat 18-22 weeks of pregnancy, obtained from the MRC/Wellcome Trust HumanDevelopmental Biology Resource upon informed consent and approval by ourlocal ethics and biozahard board committee (CMRBCEIC-26/2013). Neonataland adult tissues were obtained from the BST upon IRB approval(HCB/2018/0030). Primary T-ALL samples and diagnostic immunophenotypingdata were obtained from the local hospitals Sant Joan de Den, GermansTrias i Pujol, and Santa Creu i San Pau (Barcelona, Spain). Forimmunophenotyping of T-ALL primary samples, the followingfluorochrome-conjugated monoclonal antibodies (MoAb) were used:anti-CD2-PE, CD7-FITC/PE, CD13-PerCP-Cy5.5, CD34-APC, CD3-PE, CD5-FITC,CD4-BV-421, CD8-APC-Cy7, CD45-AmCyan, CD1 a-BV-421/APC/PE, CD33-APC andCD123-APC (BDBiosciencies or Miltenyi Biotec). Isotype-matched,non-reactive fluorochrome-conjugated MoAb were always used as afluorescence reference. Briefly, PB mononuclear cells (PBMCs, 5×10⁵)were incubated with erythrocyte-lysing solution (BDBiosciencies) for 10min and then stained with MoAb (20 min at 4° C. in the dark). Stainedcells were washed in phosphate buffered saline (PBS) and analyzed byFACS on a FACSCanto-II flow cytometer equipped with FACSDiva software(BDBiosciencies)³⁰⁻³².

In Vitro Cytotoxicity Assays and Cytokine Release Determination

Cell lines Jurkat, MOLT4 and NALM6 were purchased from DSMZ(Braunschweig, Germany) and expanded according to DSMZ recommendations.Luciferase (Luc)/GFP-expressing cells were stably generated byretroviral transduction and FACS purification of GFP+ cells³³. Targetcells (cell lines and primary T-ALL blasts) were labeled with 3 μMeFluor670 (eBioscience) and incubated with CD1a, CD22 or mock CARTs atdifferent Effector:Target (E:T) ratios for the indicated time periods.CART-mediated cytotoxicity was determined by analyzing the residualalive (7-AAD-) eFluor670+ target cells at each time point and E:T ratio.Absolute cell counts were determined using Trucount absolute count beads(BD Biosciences). Additionally, FACS-sorted CD3+CD1a− mature T-cellsfrom the PB of cortical T-ALL patients at presentation were activated,transduced with CD1a CAR and tested against their eFluor670-labeledautologous CD1a+ T-ALL blasts. The production of the pro-inflammatorycytokines IL-2, TNFα and IFNγ was measured by ELISA (Human ELISA SET, BDBiosciences) in supernatants harvested after 16 hours.

In Vivo Jurkat and T-ALL Patient-Derived Xenograft (PDX) Models

Six- to 12-week-old nonobese diabetic (NOD)-Cg-Prkdcscid Il2rgtm1Wjl/SzJ(NSG) mice (The Jackson Laboratory) were bred and housed underpathogen-free conditions in the animal facility of the BarcelonaBiomedical Research Park (PRBB). Mice were irradiated (2 Gy) andintravenously (i.v.) transplanted with 3×10⁶ Luc-GFP-expressing Jurkatcells or with 1×10⁶ primary cortical CD1a+ T-ALL blasts (primary andprimograft-expanded)³⁴. Between 1.5×10⁶ to 5×10⁶ CD1a or mock CARTs werei.v. infused 3 days later. When Luc-Jurkat cells were used, tumor burdenwas followed by bioluminescence (BLI) using the Xenogen IVIS 50 ImagingSystem (Perkin Elmer). To measure luminescence, mice received 150 mg/kgof D-luciferin intraperitoneally, and tumor burden was monitored at theindicated time points. Living Image software (Perkin Elmer) was used tovisualize and calculate total luminescence. Tumor burden of primaryT-ALL samples was followed-up by biweekly bleeding and FACS analysis.Mice were sacrificed when mock CARTs-treated animals were leukemic, andtumor burden (hHLA-ABC+hCD45+hCD1a+ graft) and CART persistence(hHLA-ABC+hCD45+hCD3+hCD1a−GFP+) was analysed in BM, PB and spleen byFACS. In re-challenge experiments, leukemia-free animals that hadreceived an infusion of CD1a CARTs 5-6 weeks before were re-infused witheither 1.5×10⁶ Luc-Jurkat cells or 1×10⁶ CD1a+ T-ALL primografts, anddisease reappearance was followed-up by BLI and FACS, as above. Allprocedures were performed in compliance with the institutional animalcare and usage committee of the PRBB (DAAM7393).

Enzyme-Linked Immunospot Assay (ELISpot)

ELISpot plates (Millipore) were coated with anti-human IFNγ antibody(1-D1K, Mabtech) and kept overnight at 4° C. Plates were then washed sixtimes with PBS containing 1% fetal calf serum and then cells from threeindependent donors were plated at 5×10⁵ to 1×10⁶ cells/well and culturedin triplicate for 20 h at 37° C. and 5% CO2. We measured IFNγ-secretingcells in response to CEF at 1 μg/mL, a peptide pool of T-cell epitopesof Cytomegalovirus (CMV), Epstein-Barr virus (EBV) and Flu and tostaphylococcal enterotoxin B (SEB) at 1 μg/mL as a positive control.Plates were then revealed with biotinylated anti-human IFNγ,streptavidin-alkaline phosphatase (Mabtech), as previouslydescribed^(35,36). The frequency of IFNγ-secreting cells was quantifiedusing ImmunoCapture and ImmunoSpot software to calculate the number ofIFNγ Spot Forming Units per 10⁵ (SFU).

Statistical Analysis

Data from at least three individual donors are shown in all figures, andexperimental duplicates were always performed. At least five animalswere used in each in vivo condition. All p-values were calculated byunpaired two-tailed Student's t-test using Prism software (GraphPad).Event-free-survival (EFS) of mice was determined using a Mantle-Coxtest. A p-value <0.05 was considered statistically significant.

Example 1: CD1a Specifically Marks Cortical T-ALL Blasts

The shared expression of target antigens between CARTs and T-lineageblasts has limited immunotherapy approaches in T-ALL due to CART-relatedfratricide and potential life-threating T-cell aplasia. However, CD1aantigen is expressed in cortical T-ALLs, a major subset of T-ALLs (FIG.1A,B), but is completely absent in functional T-cells in allextra-thymic tissues²⁵, and steady-state CD34+HSPCs lack CD1a expressionin multiple hematopoietic sites across ontogeny (FIG. 1C). T-celldevelopment is initiated within the thymus by a first colonizingCD34^(high)CD7-CD1a− primitive HSPC with lympho-myeloid potential, whichthen differentiate in response to the thymic microenvironment intoCD34^(high)CD7+CD1a− early T-cell progenitors³⁷. As they progressthrough thymic differentiation, T-cell progenitors maintain CD7expression and gradually lose CD34 expression, whereas CD1a expressionemerges and is transiently confined to cortical thymocytes³⁸ (FIG.1E,F). Within the CD34+ thymic population, ˜50% is represented bypre-cortical T-cell progenitors (CD34highCD7+CD1a−, 1 E,F (grey cells)),allowing us to hypothesize that CD1a may be a feasible and safe targetfor immunotherapy in R/R cortical T-ALL, which have a fataloutcome^(3,39-41).

Example 2: CD1a-Redirected T-Cells (CD1a CARTs) Expand without T-CellFratricide

We designed a second-generation CD1a CAR consisting of anti-CD1a scFv, aCD8 TM spacer, and intracellular signaling domains from 4-1BB (CD137)and CD3 coupled in-frame with GFP through a T2A sequence (FIG. 2A). Theexpression of the CD1a CAR was easily detected through coexpression ofboth scFv and GFP in 293T cells (FIG. 2B) and in primary CD4+ and CD8+T-cell subsets (FIG. 2C). Importantly, activated (CD69+CD25+) CD1a CARTs(FIG. 2D) continuously expanded 200-fold over a 12-day period, similarto MOCK T-cells (FIG. 2E), demonstrating that redirecting CARTs againstCD1a antigen does not induce T-cell fratricide.

Example 3: CD1a CARTs Specifically Eradicate T-ALL Cell Lines andPrimary Blasts In Vitro

CD1a CARTs were then tested in vitro using the CD1a+ T-ALL cell linesJurkat and MOLT4, and the B-ALL cell line NALM6 as a negative control(FIG. 2F). Compared with control CARTs (either MOCK T-cells or CD22CARTs), CD1a CARTs specifically eliminated CD1a+ T-ALL cells in a mannerdependent on the E:T ratio. A relatively low E:T ratio of 2:1 or 4:1induced 50-80% specific cell lysis in 16 h-assays (FIGS. 2H,I, 9).Importantly, barely any CD1a+ T-ALL cells survived exposure to CD1aCARTs in a 72 h-assay at a 1:1 E:T ratio (FIG. 21). CD1a CARTs producedhigh levels of the pro-inflammatory cytokines IL-2, TNFα and IFNγ onco-culture with CD1a+ T-ALL cells confirming their action (FIG. 2K).

To further address their ability to eliminate primary tumors, CD1a CARTswere co-cultured with primary cortical T-ALL samples (either freshlyharvested or PDX-derived), with a proportion of CD1a+ blasts rangingbetween 80% and 98% (FIG. 3A). Compared with MOCK T-cells, CD1a CARTsspecifically eliminated primary CD1a+ cortical T-ALL cells in 72 hcytotoxicity assays at 4:1 E:T ratio (FIG. 3B,C). Normal hematopoieticcells (CD1a−) co-existing in BM with CD1a+ T-ALL blasts were not lysedby CD1a CARTs (FIG. 3C). High-levels of IFNγ and TNFα were also secretedon co-culture with CD1a+ primary T-ALL cells (FIG. 3D). Collectively,these results show that CD1a CARTs have a potent and specificanti-leukemic activity against T-ALL cell lines and primary blasts invitro.

Example 4: CD1a CARTs Demonstrate Potent Anti-Leukemia Activity In Vivo

We next evaluated the activity of CD1a CARTs in vivo using bothLuc-expressing Jurkat T-ALL cells (FIG. 4, 10) and a primary corticalT-ALL xenograft model³⁴ (FIG. 5). NSG mice were transplanted with 3×10⁶Luc-expressing Jurkat cells three days prior to i.v. infusion of either2×10⁶ or 5×10⁶ CD1a (or MOCK) CARTs, and leukemia establishment wasfollowed-up weekly by BLI (FIG. 4A, 10). In contrast to the micereceiving MOCK T-cells, which showed massive tumor burden by BLI, thosemice receiving CD1a CARTs were practically leukemia-free by day 25 (FIG.4B,C, 10). The control of leukemia progression was CD1a CART celldose-dependent (FIG. 10B,C). Flow cytometry analysis of tumor burden inPB at sacrifice confirmed the BLI data (FIG. 4D). Importantly, FACSanalysis revealed T-cell persistence in all hematopoietic tissuesanalyzed (FIG. 4E); however, we found a significantly increasedbiodistribution of CD1a CARTs in BM and spleen, as compared with T-cellbiodistribution in mice receiving MOCK T-cells (FIG. 4E), indicative ofan active control of disseminated leukemia by CD1a CARTs.

In a clinically more relevant PDX model of cortical T-ALL, NSG mice werefirst transplanted with 1×10⁶ primary CD1a+ T-ALL blasts followed threedays later by infusion of 1×10⁶ CD1a (or MOCK) CARTs, and leukemiaengraftment was then followed-up bi-weekly by bleeding and endpoint BManalysis (FIG. 5A). Engraftment of CD1a+ cortical T-ALL cells graduallyincreased over time both in BM (FIG. 5B, 50%±13% and 55%±11% on week 6and 9, respectively) and PB (FIG. 5C, 4.4%±2% and 18%±6% on week 6 and9, respectively) in MOCK T-cells-treated PDXs, and associated with asignificantly lower 9-week OS (42% vs 100%, p=0.01; FIG. 5D). Incontrast, CD1a CARTs fully abolished T-ALL growth/engraftment (0.36% and0% T-ALL blasts in BM and PB, respectively) and they persisted in BM andPB after 9 weeks (FIG. 5B,C,E).

Example 5: In Vivo Persistent CD1a CARTs are Functional in Re-ChallengeAssays

Because the persistence of CARTs in hematopoietic tissues is a majorbiological parameter for their clinical success, we next assessedwhether CD1a CARTs persisting after 40-50 days remained functional andefficient in controlling T-ALL progression. To do this,T-ALL-transplanted mice in which the leukemia was abolished on treatmentwith CD1a CARTs were rechallenged with either Luc-Jurkat cells (FIG.6A-D) or primary T-ALLs from a primograft (FIG. 6E-G). In contrast tocontrols in which the secondary leukemias rapidly (as soon as 2 weeksafter) and massively engrafted, T-ALL engraftment was barely detectableby either BLI or FACS in the Jurkat (FIG. 6C) or primograft model after6 weeks (FIG. 6F).

Example 6: Patient-Derived CD1a CARTs Specifically Target AutologousCD1a+ Blasts and Retain Antiviral Activity

The proper choice of the target antigen and avoiding T-cell fratricideare crucial for the success of CARTs in the treatment of T-ALL.Accordingly, we examined whether PB-derived CD3+CD1a− T-cells frompatients with cortical T-ALL can be isolated and genetically modified toexpress CD1a CAR (FIG. 7). Thus, CD3+CD1a− T-cells from patients wereisolated (>95% purity, data not shown), activated with CD3/CD28 andlentivirally transduced (31-70% transduction) with CD1a CAR or MOCK.Next, we investigated the cytolytic capacity of CD1a CARTs derived fromprimary T-ALLs against active T-ALL patient-matched PBMCs (FIG. 7A).Total PBMCs were used as targets because it allows us to assess both theautologous cytotoxicity potential and the degree of fratricide. WithineFluor670-labelled target PBMCs, the great majority are CD1a+ blasts and−15% are CD3+CD1a− normal T-cells (FIG. 7B). As compared with MOCKT-cells, the CD1a CARTs showed a massive and specific cytolytic capacityagainst autologous CD1a+ blasts but not against CD1a− normal T-cells(FIG. 6B), further demonstrating that CD1a CARTs arefratricide-resistant.

To further assess the potential thymic toxicity of CD1a CARTs, we nextused human normal fetal thymus-derived CD7+ thymocytes as target cells.Only the CD1a+ cortical thymocytes (second and third grey box) wereeliminated by the CD1a CARTs, whereas developmentally earlier and laterCD1a− (first box) thymic T-lineage populations (CD7+CD34+ and CD7+CD34-)were not targeted (FIG. 1E,F), limiting the on-target/off-tumor effectsto a developmentally transient thymic population of cortical thymocytes.We finally sought to determine whether CD1a CARTs can protect, bythemselves, the host by targeting the most common pathogens causingviremia in immunosuppressed patients. To do this, we tested thereactivity of CD1a CARTs to CMV, EBV and Flu antigens (CEF) andquantified the SCFs by IFNγ ELISpot. Both MOCK T-cells and CD1a CARTsresponded very similarly to stimulation with viral peptides, suggestingthat CD1a CARTs retain antiviral activity (FIG. 7D).

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1. A method of treating a CD1a-positive cancer in a subject in needthereof, said method comprising administering to said subject a T cellcomprising a nucleic acid encoding a chimeric antigen receptor (CAR)comprising: (i) an extracellular domain comprising a CD1atargeting-moiety, wherein the CD1a targeting moiety is a scFV comprisinga VL domain consisting of SEQ ID NO: 7 and a VH domain consisting of SEQID NO: 8; (ii) a transmembrane domain; and (iii) an intracellularsignaling domain.
 2. The method according to claim 1, wherein thetransmembrane domain comprises the transmembrane domain of CD28, CD3,CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.
 3. The method according to claim 2, wherein thetransmembrane domain comprises the transmembrane domain of CD8.
 4. Themethod according to claim 1, wherein the intracellular signaling domaincomprises the intracellular domain of CD3ξ, FcRγ, CD3γ, CD3δ, CD3ε, CD5,CD22, CD79a, CD79b or CD66b.
 5. The method according to claim 4, whereinthe intracellular signaling domain comprises the intracellular domain ofCD3ξ.
 6. The method according to claim 1, wherein the CAR furthercomprises a costimulatory signaling domain.
 7. The method according toclaim 6, wherein the costimulatory signaling domain comprises theintracellular domain of CD27, CD28, CD137, CD134, CD30, CD40, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, CD278 orCD276.
 8. A method of treating a CD1a-positive cancer in a subject inneed thereof, said method comprising administering to said subject apharmaceutical composition comprising a plurality of T cells as definedin claim 1 and a pharmaceutically acceptable carrier or diluent.
 9. Themethod according to claim 1, wherein the CD1a-positive cancer iscortical T-cell acute lymphoblastic leukemia.
 10. The method accordingto claim 8, wherein the CD1a-positive cancer is cortical T-cell acutelymphoblastic leukemia.
 11. The method according to claim 1, wherein theCD1a-positive cancer is CD1a+ T− cell lymphoblastic lymphoma.
 12. Themethod according to claim 8, wherein the CD1a-positive cancer is CD1a+T-cell lymphoblastic.
 13. The method according to claim 9, wherein theCD1a-positive cancer is relapsed/refractory cortical T-cell acutelymphoblastic leukemia.
 14. The method according to claim 10, whereinthe CD1a-positive cancer is relapsed/refractory cortical T-cell acutelymphoblastic leukemia.
 15. The method according to claim 11, whereinthe CD1a-positive cancer is relapsed/refractory CD1a+ T-celllymphoblastic lymphoma.
 16. The method according to claim 12, whereinthe CD1a-positive cancer is relapsed/refractory CD1a+ T-celllymphoblastic lymphoma.
 17. The method according to claim 7, wherein thecostimulatory signaling domain comprises the intracellular domain ofCD137.